Viroporins are a diverse class of small hydrophobic proteins that insert into intracellular or the plasma membrane and form pores to disrupt cellular ion homeostasis. Chief among the biologically relevant ions is calcium, a ubiquitous secondary messenger that is involved in many signaling pathways. Rotaviruses (RV) are the leading cause of childhood viral gastroenteritis, resulting in diarrhea, vomiting and life-threatening dehydratio. One mechanism for the destruction of RV-infected enterocytes is the dramatic elevation in cytoplasmic calcium, which is triggered by the rotavirus nonstructural protein 4 (NSP4). Recent data show that NSP4 viroporin activity releases endoplasmic reticulum (ER) calcium stores and causes a steady-state elevation in the concentration of cytoplasmic calcium. Elevated cytoplasmic calcium is also necessary for RV replication, so NSP4 viroporin activity is likely a critical aspect of the RV replication strategy and an attractive target for inhibitors. This proposl seeks to define the functional characteristics of the NSP4 viroporin, demonstrate the importance NSP4 viroporin activity has for RV replication, and assess the ability of putative NSP4 viroporin inhibitors to reduce RV replication and pathogenesis. Aim 1 will use lipid bilayer and patch clamp electrophysiology experiments to define NSP4 ion channel activity in both artificial and native ER membranes. Putative inhibitors of NSP4 viroporin activity will be also tested for NSP4 ion channel inhibition. Aim 2 seeks to demonstrate the importance of NSP4 viroporin activity and viroporin-mediated elevation of cytoplasmic calcium for the assembly of RV replication complexes, called viroplasms. NSP4 ion channel inhibitors will be tested for their ability to block the NSP4-induced elevation in cytoplasmic calcium, and block RV replication and viroplasm assembly in RV-infected cells. Aim 3 will test the putative NSP4 inhibitors in a mouse model of RV diarrhea, to determine whether blocking NSP4 viroporin activity will protect animals from diarrhea. Completion of these studies adds to our fundamental understanding of viroporin ion channel activity, and constitutes the first electrophysiology studies on a viral calcium channel. These experiments also will determine whether inhibition of the rotavirus NSP4 viroporin effectively blocks virus replication, demonstrating that NSP4 is a good antiviral drug target. The data and techniques in these studies will form the basis of my independent research program on viral calcium channels and development of viroporin inhibitors as tools for molecular virology studies and leads for antiviral drug development. My two mentors, Dr. Frank Horrigan and Dr. Timothy Palzkill, are experts in electrophysiology and drug development, respectively. The members of my Advisory Committee each have unique expertise in areas relevant to the proposed studies. The advanced training I receive through this award will foster the development of skills and new expertise necessary for an independent biomedical research career focused on viral calcium channels, particularly those of important human pathogens. PUBLIC HEALTH RELEVANCE: These studies will characterize viral ion channels (viroporins) that disrupt cellular calcium levels, which benefits virus replication but results in host cell damage or death. Changes in cellular calcium caused by rotavirus NSP4 are likely to contribute to rotavirus-induced diarrhea and vomiting, so drugs that inhibit NSP4 ion channel function are expected to lessen the severity of rotavirus disease. Defining the functional characteristics of NSP4, which is the prototype viral calcium channel, should provide relevant information to understand possible functions of analogous ion channels produced by other pathogens [e.g., human immunodeficiency virus (HIV), hepatitis C virus (HCV), and human papillomavirus (HPC)] and guide development of effective drugs that inhibit replication of these viruses, reduce virus pathogenesis, and protect people against disease and death.